Toxicology Letters 234 (2015) 120–130
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
CDK14 expression is down-regulated by cigarette smoke in vivo and in vitro Daniel Pollack a , Yuxuan Xiao a , Vibha Shrivasatava a , Avi Levy a , Miriam Andrusier a , Jeanine D’Armiento b , Marina K. Holz a,c, Margarita Vigodner a,d, * a
Department of Biology, Stern College, Yeshiva University, New York, NY, USA Division of Pulmonary, Allergy & Critical Care Medicine, Department of Medicine, Columbia University, New York, NY, USA Department of Molecular Pharmacology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA d Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 January 2015 Accepted 8 February 2015 Available online 10 February 2015
In this study, DNA arrays have been employed to monitor gene expression patterns in testis of mice exposed to tobacco smoke for 24 weeks and compared to control animals. The results of the analysis revealed significant changes in expression of several genes that may have a role in spermatogenesis. Cdk14 was chosen for further characterization because of a suggested role in the testis and in regulation of Wnt signaling. RT-PCR analysis confirmed down regulation of Cdk14 in mice exposed to cigarette smoke (CS). Cdk14 is expressed in all testicular cells; spermatogonia- and Sertoli-derived cell lines treated with cigarette smoke extract (CSE) in vitro showed down-regulation of CDK14 mRNA and protein levels as well as down-regulation of b-catenin levels. CS-induced down-regulation of CDK14 mRNA and protein levels was also observed in several lung epithelium-derived cell lines including primary normal human bronchial epithelial cells (NHBE), suggesting that the effect is not restricted to the testis. Similar to testicular cells, CS-induced down-regulation of CDK14 in lung cells correlated with decreased levels of b-catenin, a finding suggesting impaired Wnt signaling. In the lungs, CDK14 was localized to the alveolar and bronchial epithelium. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cigarette smoke CDK14/PFTK1/PFTAIRE-1 Cyclin Y b-Catenin Spermatogenesis Spermatogonia DNA arrays Lungs Normal human bronchial cells
1. Introduction It is generally agreed that tobacco smoke can be a cause of serious health-related problems (Witschi, 2001; Doll et al., 2004). Pathologies linked to smoking tobacco cigarettes include diseases of the cardiovascular system, in particular atherosclerosis, myocardial infarction and stroke, and diseases of the respiratory tract, such as emphysema, lung cancer and cancers of the larynx and mouth. These effects are thought to stem from long term exposure to more than 3000 chemicals found in tobacco smoke, such as nicotine, carbon monoxide, cyanide, and other compounds that are believed to be responsible for various forms of tissue damage. Exposure to cigarette smoke may be either active or passive. While the association between inhalation of mainstream
* Corresponding author at: Department of Biology Stern College, Yeshiva University, 245 Lexington Avenue, New York, NY 10016, USA. Tel.: +1 212 340 7769; fax: +1 212 340 7868. E-mail address:
[email protected] (M. Vigodner). http://dx.doi.org/10.1016/j.toxlet.2015.02.006 0378-4274/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
smoke and diseases has been established for many years, the realization that exposure to second hand smoke adversely affects human health is more recent (Jinot and Bayard, 1994; Chidekel, 2000; Cook and Strachan, 1999; Strachan and Cook, 1997). Adult spermatogenesis is a multiphase process that includes proliferation of spermatogonia, meiosis of spermatocytes, and post-meiotic maturation of spermatids (spermiogenesis). Spermatogenesis is regulated by controlled expression of specific genes at different stages of germ cell maturation. The process is supported by growth factors and hormones secreted by Sertoli and Leydig somatic cells. Successful progression through spermatogenesis is crucial for normal gamete formation and for transferring the genetic information to the next generations. Several studies have shown that smoking might be associated with increased prevalence of abnormal sperm and changes in steroid hormone concentrations in humans (Attia et al., 1989; Chia et al., 1994; Field et al., 1994). A study in rats has shown a significant correlation between cigarette smoke exposure and impaired testicular histology; however, the mechanism underlying the adverse effect of tobacco on
D. Pollack et al. / Toxicology Letters 234 (2015) 120–130
testicular cells has not been well-characterized. As recently shown by our group, exposure of mice to tobacco smoke in vivo causes oxidative stress and changes in posttranslational modifications of proteins in mouse testicular cells and in human sperm (Shrivastava et al., 2010; Vigodner et al., 2013). Another study has found a significant increase in germ-line mutation frequency in spermatogonial stem cells of mice exposed to tobacco (Yauk et al., 2007). It has also been demonstrated that CSE-treated spermatocytes show signs of oxidative damage and increased expression of several antioxidant genes (Esakky et al., 2012). However, tobacco-induced changes in gene expression in vivo remain largely uncharacterized. In this study, we employed DNA arrays to examine and compare gene expression patterns in testis of mice exposed to tobacco smoke for 24 weeks as compared to control animals. We observed significant changes in several genes with a putative role in spermatogenesis, and further analyzed the effects of cigarette smoke on the expression of CDK14 (cyclin-dependent kinase 14) in multiple cell lines in vitro.
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2.3. Preparation of cigarette smoke extract CSE was prepared as described previously (Calogero et al., 2009; Mercer et al., 2009; Lawson et al., 1998; Lemaitre et al., 2011). In brief, one research cigarette (3R4F) was attached to a tube connected to a Buchner flask containing 25 mL PBS. The smoke derived from the cigarette was drawn into the flask under a vacuum generated by a nickel-plated water aspirator. The pH of the solution was then adjusted to 7.2–7.4 with 1 N HCl and filtered through a 0.22-mm pore filter to remove bacteria and large particles. The resulting 100% CSE was diluted with PBS to achieve 1–10% concentrations and used within 30 min of preparation. The concentrations of 1–10% CSE correspond to the nicotine concentrations in the extract which is similar to these measured in the blood of the smokers (Calogero et al., 2009; Lawson et al., 1998). This concentration range was also commonly used for cell treatment in other previously published studies (Mercer et al., 2009; Lemaitre et al., 2011). Treatments for the indicated time periods were followed by preparation of whole-cell protein lysates. Each experiment was repeated at least three times.
2. Materials and methods 2.4. Gene array and statistical analysis 2.1. Tobacco exposure Adult (6–8 week old) C57B16 male mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Animal Committee of Yeshiva University approved all animal protocols. The tobacco smoke exposure was performed in the laboratory of Dr. Jeanine D’Armiento, Division of Molecular Medicine, Columbia University, New York as previously described (Foronjy et al., 2006). After acclimatization for three days, five mice were exposed to cigarette smoke in a specially designed chamber for 6 h/day 5 days a week for 24 weeks at a total particulate matter (TPM) concentration of 250 mg/m3. The TPM was determined by gravimetric analysis of filter samples taken during the exposure periods. Two 70 mL puffs per minute from research cigarettes (Type 3RF; 3R4F: 10 mg of tar and 0.8 mg of nicotine, Tobacco Research, University of Kentucky, Lexington, KY, USA) were generated by a smoking machine (Teague Enterprises, Davis, CA) and then diluted with fresh air and delivered to whole body exposure chambers. Mice were provided with standard food and water ad libitum and maintained at room temperature in a 12-h dark/light cycle. Mice showed no signs of adverse effects or abnormal behavior during or after the smoke exposure. Five control mice were exposed to room air. The same exposure schedule has previously been used in several studies from D’Armiento’s laboratory and in a previously published study from our group showing an adverse effect of CS on testicular cells (Shrivastava et al., 2010). 2.2. Cell lines GC1 spermatogonia and 15P1 Sertoli were obtained from American Type Culture Collection (ATCC). Cells were cultured in DMEM supplemented with 10% fetal bovine serum, incubated at 37 C (5% CO2). Immortalized human bronchial epithelial cells (HBEC), a gift from Dr. Spivack (Albert Einstein College of Medicine), were cultured in keratinocyte serum-free medium (Life technologies, Cat# 17005-042) containing 50 mg/L bovine pituitary extract with 5 mg/L epidermal growth factor (Tan et al., 2010). Human bronchial epithelial cells (BEAS-2B) were purchased from ATCC (Cat# CRL-9609TM) and maintained in bronchial epithelial cell growth medium (BEGM, Cat# CC-3170) containing Clonetics1 bronchial epithelial cell basal medium with supplements provided by Lonza. Normal human bronchial epithelial cells (NHBE) were purchased from Lonza (Cat# CC-2540) and maintained in BEGM produced by Lonza.
Testes of control and cigarette smoke-exposed mice were obtained, and RNA was isolated from the samples using the RNeasy mini kit (Qiagen). RNA integrity was tested by microfluidic analysis using the Agilent 2100 BioAnalyzer. The microarray analysis was performed at the Albert Einstein College of Medicine of Yeshiva University Microarray Facility. For each sample, the Affymetrix whole-transcript protocol was used to amplify 300 ng RNA and hybridized to the Affymetrix Mouse Gene 1.0 ST Array. Three mice were used per condition (control and cigarette smoke) to provide biological triplicates. Images of each array have been captured using the Axon scanner, and the pixel intensities from each channel of individual features on the chip were determined using the Genepix 3.0 program. The raw array data were imported into Expression Console v1.1 (Affymetrix), a software package that permits visualization, quality control and normalization of the data. Quality control (QC) results included array images, line graphs of labeling and hybridization controls, signal box plots and histograms before and after normalization for all hybridizations, as well as a heatmap for Spearman rank correlation between all pairs of hybridizations. The gene expression measures for all arrays that passed QC were then normalized by the robust multi-array analysis (RMA) approach in the Expression Console software package. Ranking of genes by degree of differential expression was performed using Bioconductor package limma and in-house R code. Selection of significantly different gene expression profiles between the two experimental conditions was based on the empirical Bayes moderated t-statistic and Benjamini–Hochberg method was applied to correct for multiple testing. Significant genes were identified by adjusted-P value 0.05 and folddifference in mean expression |1.4|. All results are expressed as the mean standard deviation (SD) unless otherwise stated.
2.5. Real time RT-PCR Total RNA was extracted from testicular tissues using the RNeasy Mini Kit (Qiagen, Valencia, CA). The quality and concentration of RNA in samples was determined using Nanodrop 2000 (Thermo Fisher Scientific, West Palm Beach, FL). Equal amounts of RNA (500 ng) from different samples were used for cDNA synthesis using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions.
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D. Pollack et al. / Toxicology Letters 234 (2015) 120–130
Table 1 Top genes show statistically significant changes after tobacco exposure versus control at 24 weeks. Affyid
Symbol
10390211
140486 0.62 7.69
23915 10566201
Igf2bp1 Insulin-like growth factor 2 mRNA binding protein 1 Cdk14 Cyclin-dependent kinase 14 Ccdc80 Coiled-coil domain containing 80 Ankfn1 Ankyrin-repeat and fibronectin type III Olfr589 Olfactory receptor (OR)589
18835 10521498
Crmp1
4687
19465 10527940 9672 10435948 4647 10389759
Name
Entrez
LogFC AveExpr Adj.P. Val
B
Comments
0.02
5.41
RNA-binding, IFG signaling
18647 0.96 8.35 67896 0.71 6.23 382543 0.56 5.52
0.02 0.02 0.02
4.62 Regulation of mitosis and meiosis 4.37 Glucose, lipid metabolism; peroxisomal 4.22 Membrane protein
259054 0.47 5.05
0.02
4.16
12933
0.73 7.45
0.02
OR receptors may function as a chemosensing receptors in mouse germ cells and sperm 3.76 Expressed in spermatids
74062
1.48
7.82
0.02
3.66 A trascribed pseudogene; testis-specific.
9.77
0.02
3.3
Collapsin response mediator protein 1 18692 10519811 Speer8- Spermatogenesis associated ps1 glutamate (E) 8111 10420286 Gzmn Granzyme N
245839 0.4
4631
Lactoperoxidase
76113
0.54 6.34
0.03
Recombination activating gene 1
19373
0.45 6.03
0.03
10389639 Lpo
14980 10485372 Rag1
Expressed in spermatids and spermatocytes, may be involved in spermatogenesis 3.07 May have a role in oxidative stress in testicular cell mitochnodria 2.97 Immunoglobulin; may also be produced by testicular cells
LogFC: log2 fold change for that gene. A positive value indicates up-regulation of a gene, a negative value indicates down-regulation; aveExpr: average expression value for that gene across all the arrays; adj.P.Val: adjusted p-value (a p-value adjusted for multiple testing by Benjamini–Hochberg method); B: the log odds that the gene is differentially expressed. A B-statistic of zero corresponds to a 50–50 chance that the gene is differentially expressed.
The primers used, based on the cDNA sequences were as follows: Mouse primers: Cdk14 forward: 50 -CTGCGAGGACTGTCTTACATC-30 ; Cdk14 reverse: 50 -TGGCTAGGGACGGATTTTG-30 Gapdh forward: 50 -CTTTGTCAAGCTCATTTCCTGG-30 ; Gapdh reverse: 50 -TCTTGCTCAGTGTCCTTGC-30 Human primers: CDK14 forward: 50 -TGTCAGTACATGGACAAGCACCCT-30 ; CDK14 reverse: 50 -TGTAAGACAGACCTCGCAGCAACT-30 GAPDH forward: 50 -GCAAGAGCACAAGAGGAAG-30 ; GAPDH reverse: 50 -TCTACATGGCAACTGTGAGG-30 qPCR was performed in triplicate using iQTM SYBR1 green supermix and CFX96TM Real-time system (Bio-Rad Hercules, CA). Using the Bio-Rad CFX Manager analyzing program, the values of the relative quantities (RQs) of CDK14 were calculated from their quantification cycle (Cq) measurements and then normalized to the geometric mean of the GAPDH RQs to produce the normalized expression values. For cell line experiments, controls (untreated samples) were considered as 1 and other samples were normalized to the controls. To calculate the difference between samples, the experiments were performed at least three times for each condition. Student’s paired t-test was used. P values
